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Biomedical subjects

B Arcangioli

Publications and source records attributed to B Arcangioli.

9 recordsLinked to original sources
PubMed1991

A novel switch-activating site (SAS1) and its cognate binding factor (SAP1) required for efficient mat1 switching in Schizosaccharomyces pombe.

The pattern of parental DNA strand inheritance at the mating type locus (mat1) determines the pattern of mat1 switching in a cell lineage by regulating the formation of the site-specific double-stranded break (DSB) required for mating type interconversion in Schizosaccharomyces pombe. To study the molecular basis of this programmable cell type change, we conducted structural and functional analyses of the DNA sequence flanking the DSB at mat1. We have identified and characterized a DNA-binding activity that interacts with a specific sequence located 140 bp from the DSB site. Deletion analysis of DNA sequences located distal to mat1 cassette revealed the presence of at least two switch-activating sites (SAS1 and SAS2), both of which are required for generating an efficient level of DSBs and consequently, for efficient switching. We found that SAS1 overlaps with the target site of the DNA-binding activity called SAP1 (for switch-activating protein). Point mutations generated in the SAS1 element that adversely affect binding of SAP1 protein in vitro were found to reduce the efficiency of switching in vivo, suggesting the requirement of SAP1 for switching. Pedigree analysis revealed that SAS1 is equally required for initial switching (one switch in four grand-daughters of a cell) and for consecutive switching (where the sister of a recently switched cell switches again), indicating that the two developmentally asymmetric cell divisions required to generate a particular pattern of switching share the same molecular control mechanism.

Base Sequence↗
PubMed1989

Preparation and use of a universal primed Sepharose for the purification of DNA-binding proteins.

We have devised a novel method for the construction of a DNA affinity matrix and tested its use in the purification of a sequence-specific DNA-binding protein from the yeast Saccharomyces cerevisiae. The matrix was prepared in two steps: first, a palindromic oligonucleotide containing an XhoI cohesive end was covalently linked via its loop to a Sepharose matrix; second, directly to this 'universal' primed Sepharose was ligated a 37-bp oligonucleotide, with XhoI cohesive ends, containing the sequence of the upstream activation sequence 1 (UAS1) site of the yeast iso-1-cytochrome c (CYC1) gene. After fractionating a yeast crude extract through DEAE-cellulose, heparin ultrogel and Mono Q columns, a single pass through the affinity matrix allowed the purification to apparent homogeneity of the 120-kDa protein factor P, which is responsible for the binding to the UAS1 site.

Base Sequence↗
PubMed1989

A point mutation in the CYC1 UAS1 creates a new combination of regulatory elements that activate transcription synergistically.

Dissection of the upstream activation site 1 (UAS1) of the yeast CYC1 gene showed that the A and B regions respond individually to regulation by the HAP1 protein, and that a point mutation in the B region converts this region to a translation upstream factor (TUF)-regulated element. Combinatorial analyses revealed that the transacting factors involved with these wild-type and mutant UAS1 target sites combine to activate transcription in a synergistic manner. Furthermore, combinations of heterologous factors, made possible by the point mutation, create a new specificity of regulation that differs from regulation by any one factor individually.

Base Sequence↗
PubMed1989

Functional analysis of the papilloma virus E2 trans-activator in Saccharomyces cerevisiae.

The papilloma virus E2 transcriptional trans-activator is representative of a class of transcriptional modulators that activate transcription through direct binding to cis-acting DNA sequences. In this study we measured the capacity for this mammalian virus factor to function in Saccharomyces cerevisiae. When expressed in the yeast, the bovine papilloma virus E2 trans-activator could stimulate transcription from a yeast promoter having E2 DNA-binding sites present in cis. Whereas a single E2 DNA-binding site was sufficient for trans-activation, a strong cooperative effect was observed with two E2 DNA-binding sites. The level of trans-activation was dependent on the position of the E2 DNA-binding sites in relation to the yeast promoter, with the maximal effect demonstrated when the binding sites were positioned upstream. Deleted E2 proteins, lacking part of the trans-activation or DNA-binding domains, failed to activate transcription in yeast, similar to their behavior in mammalian cells. Replacement of the amino-terminal region of the E2 trans-activation domain with a synthetic amphipathic helix partially restored the trans-activation function; however, it did not result in a molecule that exhibited cooperativity between neighboring E2 DNA-binding sites.

DNA-Binding Proteins↗
PubMed1987

Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene.

We show that the yeast HAP1 activator locus encodes a protein that binds in vitro to the upstream activation site, UAS1, of the CYC1 gene (iso-1-cytochrome c). Binding of wild-type HAP1 and truncated HAP1 derivatives to UAS1 is evident in crudely fractionated yeast extracts using the gel electrophoresis DNA binding assay. The binding of HAP1 in vitro, like the activity of UAS1 in vivo, is stimulated by heme. HAP1 binds to region B, one of two portions of UAS1 shown to be important by genetic analysis of the site. Surprisingly, HAP1 binds to the same sequence as a second factor, RC2. Both HAP1 and RC2 bind to the same side of the helix, and make similar but not identical major and minor groove contacts that span two full turns. An additional factor that binds to the second important part of UAS1, the region A factor (RAF), is also identified. A model depicting the interplay of HAP1, RC2, and RAF in the control of UAS1 is presented.

Base Sequence↗
PubMed1986

Structural features of the DNA template required for transcription in vitro by yeast RNA polymerase B (II).

Yeast RNA polymerase II initiates in vitro transcription at two sites located within the vector DNA and the cloned promoter, on a recombinant plasmid DNA containing the yeast iso1 cytochrome c promoter. Both initiation sites are found within a DNA fragment hypersensitive to osmium tetroxide modification. Using a series of yeast iso1 cytochrome c promoter deletions, we have characterized an upstream DNA sequence required for optimal transcription from this site and shown in this case a correlation between osmium sensitivity and the capacity of RNA polymerase to initiate. However, perturbation of the double helix is not sufficient to generate a transcription initiation site. Insertion of 28 alternating AT residues at the EcoRV site of pBR322 generates an site hypersensitive to osmium tetroxide modification, that does not serve as a transcription start site.

Binding Sites↗
PubMed1986

A single Saccharomyces cerevisiae upstream activation site (UAS1) has two distinct regions essential for its activity.

Several site-directed mutagenesis regimens were used to generate single- and multiple-base substitutions in the upstream activation site UAS1 of the Saccharomyces cerevisiae CYC1 gene. Mutations resulting in large reductions in activity of the site lie in two distinct regions. Six single-base changes in a region A, between -288 and -285, all resulted in a 15-fold reduction in activity. Synthetic sites built up solely of multimers of the -289 to -285 sequence ACCGA behaved as carbon catabolite-sensitive UASs. In addition, substitution mutations in a second region, at nucleotides -266 and -265, virtually eliminated UAS1 activity. These mutations abolished the binding of a heme-dependent protein factor in vitro. Thus, UAS1 contains two essential regions both of which are required for its activity.

Base Sequence↗
PubMed1985

Identification of proteins involved in the regulation of yeast iso- 1-cytochrome C expression by oxygen.

On the basis of a gel electrophoresis retardation assay, protein(s) which interact specifically with the upstream activating site (UASc) of the yeast iso-1-cytochrome C (CYC1) gene were identified and separated by heparin ultrogel chromatography. DNase I protection experiments indicate that these factors protect a 23-bp sequence overlapping the UASc site previously defined. The specific binding activity is strongly reduced in extracts prepared from a wild-type strain grown anaerobically. It is absent in a mutant strain blocked in the biosynthesis of heme but it is restored upon the addition of the missing precursor, delta amino levulinic acid (dALA) to the growth medium. In contrast, the binding activity does not differ significantly in extracts form a wild-type strain grown in either glucose or glycerol as carbon source. These data strongly argue that the CYC1 UAS binding protein(s) that we have identified mediate the oxygen and heme control of cytochrome C biosynthesis.

Anaerobiosis↗